Bzip type transcription factors regulating the expression of rice storage protein

ABSTRACT

cDNAs (RISBZ1, RISBZ4, and RISBZ5) encoding bZIP transcription factors were isolated from a cDNA library originating in rice plant seed. The cDNAs encode novel proteins and have binding activity to the GCN4 motif. Among them, RISBZ1 activated transcription mediated by the GCN4 motif by 100-fold or more. Since the expression of RISBZ1 precedes the expression of a seed storage protein gene and is expressed only in maturing seeds, it is suggested that RISBZ1 controls the expression of rice seed storage proteins. In addition, by linking the recognition sequence of the transcription factor, the GCN4 motif, in tandem and introducing it into the promoter for a gene encoding seed storage protein to facilitate its binding to the transcription factor RISBZ1, expression of a foreign gene under the control of the modified promoters is greatly enhanced.

TECHNICAL FIELD

The present invention relates to a novel transcription factor and itsuse pertaining to the endosperm-specific expression of the storageprotein in the rice plant seed.

BACKGROUND ART

Seed storage protein is expressed in seeds only during the maturingstage, and the expression of genes encoding this protein is analyzed asa suitable model for investigating the transcription regulatorymechanism of plant genes (Goldberg, R. B. et al., Science 266: 605–614,1994). The expression of a gene that codes for a seed storage protein isknown to be regulated by the cooperation of a plurality of cis factorsin a promoter. The binding of a transcription factor to a specific cisregulatory factor is important in the initiation of transcription andthe tissue- and time-specific expression. It can be explained that theexpression of a seed storage protein is induced by several types of cisregulatory factors relating to the regulation of seed-specificexpression when transcription factors that recognize specific cisregulatory factor bind and aggregate. Functional analyses of cisregulatory factors and transcription factors of crop storage proteingenes have been conducted in order to elucidate the molecular mechanismof the expression of seed storage proteins (Thomas, T. L., Plant Cell 5:1401–1410, 1993; Morton, R. L. et al., in Seed Development andGermination, pp. 103–138, Marcel Dekker, Inc., 1995).

However, despite considerable research, analyses using transformedplants failed to identify the cis regulatory factors essential for geneexpression regulation in nearly all crops studied, and the geneexpression regulatory mechanism has still not been clearly understood.In the case of monocotyledons in particular, the promoter analyses usingstable transformed plants has been performed in only the seed storageprotein, glutelin, of the rice plants. On the other hand, in the case ofmaize, wheat and barley, analyses have been conducted using particleguns or tobacco transformants (Muller, M. and Knudsen, S., Plant J.6:343–355, 1993; Albani, D. et al., Plant Cell 9: 171–184, 1997;Marzabal, P. M. et al., Plant J. 16: 41–52, 1998).

It has been shown that the endosperm-specific expression of the seedstorage protein gene of grains is controlled by the collaborative actionof several types of cis regulatory factors. The Prolamin box (TGTAAAG),GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGT motif, whichare conserved in the seed storage protein gene promoters of numerousgrains, have been characterized as cis regulatory factors involved inendosperm-specific expression by loss-of-function and gain-of-functionanalyses (Morton, R. L. et al., In: Seed Development and Germination,pp. 103–138, Marcel Dekker Inc., 1995).

The GCN4 motif has been frequently found not only from seed storageprotein gene, but also from promoters of genes involved in themetabolism (Muller, M. and Knudsen, S., Plant J. 6: 343–355, 1993).Recently, a polymer of the GCN4 motif of rice plant glutelin gene hasbeen found to reproduce endosperm-specific expression in transformedrice plants, and remarkable decrease in promoter activity and changes inits expression pattern have been found due to the substitution ordeletion of nucleotides in the GCN4 motif. These facts prove that theGCN4 motif plays an important role in endosperm-specific expression (Wu,C. Y. et al., Plant J. 14: 673–683, 1998). The GCN4 motif is coupled toa Prolamin box (TGTAAAG) via a plurality of bases in many cases, and isone of the constituents of the two-factor endosperm box found in theprolamin gene promoters of nearly all grains, including wheat glutenin,barley hordein, rye secalin, sorghum cafulin and adlay coixin. The AACAmotif is involved in the expression of nearly all rice glutelin genes.Although the combination of two motifs (GCN4 motif and Prolamin box orGCN4 motif and AACA motif) is required for gene expression, in order toadequately function as an endosperm-specific promoter, an additionalmotif is essential (Takaiwa, F. et al., Plant Mol. Biol. 30: 1207–1221,1996; Yoshihara, T. et al., FEBS Letts. 383: 213–218, 1996; Wu, C. Y. etal., Plant J. (in press)). Recently, it has been demonstrated that, inorder to function as aminimum promoter capable of reproducingendosperm-specific expression in glutelin genes (GluB1) of rice plant,at least three constituents, the GCN4 motif, the AACA motif, and theACGT motif, present in the −197 bp promoter region, are essential (Wu,C. Y. et al., Plant J. 14: 673–683, 1998; Wu, C. Y. et al., Plant J. 23:415–421, 2000).

Opaque2 (O2) of maize is an endosperm-specific transcription factor ofthe bZIP type, and this O2 binds to the ACGT motif in the 22 kDa α-zeingene promoter of maize to activate transcription (Schmidt, R. J. et al.,Plant Cell 4: 689–700, 1992). O2 has been reported to be involved inendosperm-specific transcription of b-32 ribosome deactivating proteingene by binding to the (Ga/tTGAPyPuTGPu) sequence (Lohmer, S. et al.,EMBO J. 10: 617–624, 1991). O2 is thus considered to have a wide-rangingbinding capability. Reportedly, the GCN4 motif is recognized by O2, andtranscription is activated through the binding of O2 to the GCN4 motif(Wu, C. Y. et al., Plant J. 14: 673–683, 1998; Holdsworth, M. J. et al.,Plant Mol. Biol. 29: 711–720, 1995). In seeds, during the maturingstage, in vivo footprint analysis showed that the nuclear protein bindsto the GCN4 motif and Prolamin box present in wheat low molecular weightglutenin gene promoter (Vicente-Carbajos, J. et al., Plant J. 13:629–640, 1998) and maize γ-zein gene promoter (Marzabal, P. M. et al.,Plant J. 16: 41–52, 1998). In addition, the results of an in vitroDNaseI footprint analysis showed that the nuclear protein of maturingrice plant seeds as well as GST-O2 fused protein specifically recognizethe GCN4 motif of the rice glutelin gene promoter (Wu, C. Y., et al.,Plant J. 14: 673–683, 1998; Kim, S. Y. and Wu, R., Nucl. Acids Res. 18:6845–6852, 1990). These findings indicate that an O2-like transcriptionfactor is present in grain seeds, and that it controls theendosperm-specific expression of numerous seed storage protein genesmediated by the GCN4 motif.

Recently, cDNA clones of transcription factors that recognize the GCN4motif have been isolated in wheat (Albani, D. et al., Plant Cell 9:171–184, 1997) and barley (Vicente-Carbajos, J. et al., Plant J. 13:629–640, 1998; Onate, L. et al., J. Biol. Chem. 274: 9175–9182, 1999),and have been named SPA, BLZ1 and BLZ2. These transcription factors havebeen determined to activate the transcription of seed storage proteingenes mediated by the GCN4 motif in wheat low molecular weight gluteninand barley B1 hordein gene promoter. Interestingly, these transcriptionfactors were expressed seed-specifically. Although cDNA that codes for atranscription factor having a high homology with the bZIP domain of O2has previously been isolated from rice plants, it remains to beconfirmed whether or not it activates transcription of seed storageprotein gene mediated by the GCN4 motif (Izawa, T. et al., Plant Cell 6:1277–1287, 1994; Nakase, M. et al., Plant Mol. Biol. 33: 513–522, 1997).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel transcriptionfactor that regulates the expression of rice seed storage protein bybinding to the GCN4 motif, a gene that codes for the factor, plant cellsand plant bodies in which the gene has been introduced, and a method forproduction and use thereof.

The present inventors conducted research to resolve the above problems.As mentioned above, the GCN4 motif is a sequence that is highlyconserved in the promoters of grain seed storage protein genes, andplays a central role in the endosperm-specific expression of the genes.This GCN4 motif is recognized by the bZIP transcription factor familythat is closely related to the Opaque2 (O2) protein of maize. Therefore,the present inventors thought that, by isolating bZIP transcriptionfactor from the rice seeds, it would be possible to identify thetranscription factor that binds to the GCN4 motif to control theexpression of rice seed storage protein.

First, the present inventors screened a cDNA library originating in riceseed and isolated cDNA that codes for five types of bZIP transcriptionfactors (RISBZ1, RISBZ2, RISBZ3, RISBZ4, and RISBZ5). Based on thehomology of the presumed amino acid sequences, RISBZ2 and RISBZ3 wereidentical to RITA1 (Izawa, T. et al., Plant Cell 6: 1277–1287, 1994) andREB (Nakase, M. et al., Plant Mol. Biol. 33: 513–522, 1997),respectively, and the remaining RISBZ1, RISBZ4; and RISBZ5 were revealedto code for novel proteins. When the binding ability of RISBZ1, RISBZ2,RISBZ3, RISBZ4, and RISBZ5 to GCN4 motif was investigated, they allexhibited binding activity to the GCN4 motif. Furthermore, thetranscription activation ability of the five proteins by binding to theGCN4 motif was investigated. As a result, only RISBZ1 activatedtranscription 100-fold or more by binding to the GCN4 motif. Inaddition, an analysis using the GAL4 DNA binding domain of yeastrevealed that proline-rich, 27 amino acid residues of the N-terminalside of RISBZ1 functioned as a the transcription-activating domain. Thedifference in transcription activation ability between RISBZ1 and theother RISBZ proteins was primarily due to the mutation of 7 amino acidresidues (for RISBZ2) or deletion of the transcription-activating domain(for RISBZ3, RISBZ4, and RISBZ5). This finding suggests that thedifference in transcription activation ability between RISBZ1 and otherRISBZ proteins occur due to a structural mutation of the transcriptionactivating domain. In addition, RISBZ1 was found to form not only ahomodimer, but also heterodimers with other RISBZ proteins. Since theexpression of RISBZ1 precedes the expression of seed storage proteingene and is expressed only in maturing seeds, RISBZ1 may control theexpression of seed storage protein. In order to investigate theexpression of RISBZ1 gene, the promoter of the RISBZ1 gene was coupledto a GUS reporter gene, and this construct was introduced into a riceplant. In this rice plant the GUS gene was strongly expressed in thealeurone layer.

As described above, the present inventors demonstrated that the novelproteins RISBZ1, RUSBZ4, and RISBZ5 actually bind to the GCN4 motif, andclarified that RISBZ1 is a transcription activation factor involved inendosperm-specific expression of the rice seed storage protein gene.

The present inventors also produced a transformed plant that contained aDNA construct in which the RISBZ1 of the present invention was connecteddownstream of a promoter and a DNA construct in which a reporter genewas connected downstream of a promoter containing the target sequence ofRISBZ1. The inventors then succeeded in measuring the transcriptionactivity of RISBZ1 in the transformed plant by using the expression ofthe reporter gene as an indicator. These findings enable high levelexpression of a useful, highly value-added foreign gene within thetransformed plant cells in which the foreign gene is connecteddownstream of a promoter containing the target sequence of RISBZ1instead of the above reporter gene.

The present invention relates to a novel transcription factor thatregulates the expression of rice seed storage protein by binding to theGCN4 motif, a gene encoding the factor, plant cells and plants in whichthe gene has been introduced, and methods for production and usethereof. More specifically, the present invention provides thefollowing:

[1] a DNA selected from the following (a) through (d):

-   -   (a) a DNA encoding a protein comprising the amino acid sequence        set forth in any one of SEQ ID NOs: 2, 5, and 7;    -   (b) a DNA comprising a coding region of the nucleotide sequence        set forth in any one of SEQ ID NOs: 1, 3, 4, and 6;    -   (c) a DNA comprising the amino acid sequence set forth in any        one of SEQ ID NOs: 2, 5, and 7, in which one or more amino acids        are substituted, deleted, added, and/or inserted, and encoding a        protein that is functionally equivalent to a protein comprising        the amino acid sequence set forth in any one of SEQ ID NOs: 2,        5, and 7; and    -   (d) a DNA hybridizing under stringent conditions with a DNA        comprising the nucleotide sequence set forth in any one of SEQ        ID NOs: 1, 3, 4, and 6, and encoding a protein functionally        equivalent to a protein comprising the amino acid sequence set        forth in any one of SEQ ID NOs: 2, 5, and 7;

[2] the DNA according to [1], which encodes a protein that binds to theGCN4 motif or activates expression of rice seed storage protein;

[3] the DNA according to [1] or [2], which is derived from rice plant;

[4] a DNA encoding antisense RNA complementary to a transcriptionproduct of the DNA according to any one of [1] through [3];

[5] a DNA encoding an RNA having ribozyme activity that specificallycleaves a transcription product of the DNA according to any one of [1]through [3];

[6] a DNA encoding an RNA that suppresses the expression of the DNAaccording to any one of [1] through [3] in plant cells by co-inhibitioneffects, and having 90% or more homology with the DNA according to anyone of [1] through [3];

[7] a DNA encoding a protein having a dominant negative phenotype of aprotein encoded by the DNA according to any one of [1] through [3] whichis endogenous in plant cells;

[8] a vector containing the DNA according to any one of [1] through [3];

[9] a transformed cell retaining the DNA according to any one of [1]through [3] or the vector according to [8];

[10] a protein that is encoded by the DNA according to any one of [1]through [3];

[11] a method of producing the protein according to [10], the methodcomprising steps of culturing the transformed cell according to [9] andcollecting the expressed protein from said transformed cell or theirculture supernatant;

[12] a vector containing the DNA according to any one of [4] through[7];

[13] a transformed plant cell retaining the DNA according to any one of[1] through [7] or the vector according to [8] or [12];

[14] a transformed plant containing the transformed plant cell accordingto [13];

[15] a transformed plant that is a progeny or clone of the transformedplant according to [14];

[16] a reproductive material of the transformed plant according to [14]or [15];

[17] an antibody that binds to the protein according to [10];

[18] a plant having on its genome a DNA construct in which the DNAaccording to [1] is operably connected downstream of an expressioncontrol region and a DNA construct in which a foreign gene is operablyconnected downstream of an expression control region having the targetsequence of the protein according to [10];

[19] the plant according to [18], wherein the target sequence is asequence containing the GCN4 motif;

[20] the plant according to [19], wherein the GCN4 motif has thesequence set forth in any one of SEQ ID NOs: 8, 13, and 14;

[21] the plant according to [18], wherein the target sequence is asequence containing a G/C box; and,

[22] a method of producing the plant according to any one of [18]through [21], the method comprising a step of crossing a plant having onits genome a DNA construct in which the DNA according to [1] is operablyconnected downstream of an expression control region, with a planthaving on its genome a DNA construct in which a foreign gene is operablyconnected downstream of an expression control region containing thetarget sequence of the protein according to [10].

The present invention provides DNAs encoding RISBZ1, RISBZ4, and RISBZ5protein originating in the rice plant. The nucleotide sequence of thecDNA of RISBZ1 is shown in SEQ ID NO: 1, the amino acid sequence of theprotein encoded by the cDNA is shown in SEQ ID NO: 2, and the nucleotidesequence of the genome DNA is shown in SEQ ID NO: 3 (the genome DNAsequence set forth in SEQ ID NO: 3 contains introns and is composed ofsix exons). The nucleotide sequences of the cDNAs of RISBZ4 and RISBZ5proteins are shown in SEQ ID NO: 4 and 6, respectively, while the aminoacid sequences of the proteins encoded by the cDNAs of RISBZ4 and RISBZ5proteins are shown in SEQ ID NO: 5 and 7, respectively. In the presentspecification, the RISBZ1, RISBZ4, and RISBZ5 of the present inventionare collectively referred to as RISBZ.

The RISBZ proteins of the present invention are thought to be bZIPtranscription factors having the ability to bind the GCN4 motif. Amongthese, RISBZ1 remarkably activates transcription by binding to the GCN4motif. Since the promoter of the RISBZ1 gene is activated in thealeurone layer of rice seeds, RISBZ1 is thought to be atranscription-activating factor that controls the expression of riceseed storage protein.

In addition, it has been reported that bZIP transcription factors formvarious homo/heterodimers through the combination of various factorsbelonging to the bZIP transcription factor family. As a result, controlfactors with various functions are formed, which control genetranscription. In the Examples described below, RISBZ2 and RISBZ3 wereshown to form a heterodimer with RISBZ1. In addition, RISBZ4 and RISBZ5have extremely high homology (96% and 82.7%, respectively) with the bZIPdomain of RISBZ3, and these factors would also form heterodimers withRISBZ1. These facts suggest that RISBZ4 and RISBZ5 of the presentinvention would form, with the RISBZ1 and other RISBZ members of thepresent invention, heterodimers having various transcription activatingabilities and DNA binding properties depending on the maturation stageand tissue to control the expression of seed storage protein.

Thus, the DNA encoding the RISBZ protein of the present invention, or amolecule that controls the expression of the DNA, would be useful in,for example, regulating the expression of seed storage protein.Regulation of the expression of seed storage protein has variousindustrial advantages. For example, it would be possible to accumulateabundant foreign gene products in the endosperm by deleting seed storageprotein in the endosperm. On the other hand, by highly accumulating seedstorage protein in the endosperm, it would be possible to produce seeds(e.g., rice) having greater nutritional value.

The DNA encoding the RISBZ protein of the present invention includesgenomic DNA, cDNA, and chemically synthesized DNA. A genomic DNA andcDNA can be prepared according to conventional methods known to thoseskilled in the art. More specifically, a genomic DNA can be prepared,for example, as follows: (1) extracting genomic DNA from plant cells ortissues; (2) constructing a genomic library (utilizing a vector, such asplasmid, phage, cosmid, BAC, PAC, and so on); (3) spreading the library;and (4) conducting colony hybridization or plaque hybridization using aprobe prepared based on the DNA encoding the protein of the presentinvention (e.g. SEQ ID NO: 1, 3, 4, or 6). Alternatively, a genomic DNAcan be prepared by PCR, using primers specific to the DNA encoding theprotein of the present invention (e.g. SEQ ID NO: 1, 3, 4, or 6). On theother hand, cDNA can be prepared, for example, as follows: (1)synthesizing cDNAs based on mRNAs extracted from plant cells or tissues;(2) preparing a cDNA library by inserting the synthesized cDNA intovectors, such as λZAP; (3) spreading the cDNA library; and (4)conducting colony hybridization or plaque hybridization as describedabove. Alternatively, cDNA can also be prepared by PCR.

The present invention includes DNAs encoding proteins functionallyequivalent to the RISBZ protein of SEQ ID NO: 2, 5, or 7. Herein, theterm “functionally equivalent to the RISBZ protein” means that theobject protein has the biological function equivalent to those of RISBZprotein of SEQ ID NO: 2, 5, or 7, such as the function of binding toGCN4 motif and/or regulating the expression of rice seed storageproteins. The rice seed storage proteins include, for example, riceglutelins.

Examples of such DNAs include those encoding mutants, derivatives,alleles, variants, and homologues comprising the amino acid sequence of.SEQ ID NO: 2, 5, or 7 wherein one or more amino acids are substituted,deleted, added, and/or inserted.

Examples of methods for preparing a DNA encoding a protein comprisingaltered amino acids well known to those skilled in the art include thesite-directed mutagenesis (Kramer, W. and Fritz, H. -J.,Oligonucleotide-directed construction of mutagenesis via gapped duplexDNA. Methods in Enzymology, 154: 350–367, 1987). The amino acid sequenceof a protein may also be mutated spontaneously due to the mutation of anucleotide sequence. A DNA encoding proteins having the amino acidsequence of a natural RISBZ protein (SEQ ID NOs: 2, 5, or 7) wherein oneor more amino acids are substituted, deleted, and/or added are alsoincluded in the DNA of the present invention, so long as they encode aprotein functionally equivalent to the natural RISBZ protein.Additionally, nucleotide sequence mutants that do not give rise to aminoacid sequence changes in the protein (degeneracy mutants) are alsoincluded in the DNA of the present invention. The numbers of nucleotidemutations in the object DNA at amino acid level is typically 100 aminoacids or less, preferably 50 amino acids or less, more preferably 20amino acids or less, and most preferably 10 amino acids or less (forexample, amino acids or less or 3 amino acids or less).

Whether or not a certain DNA codes for a protein having the function ofbinding to the GCN4 motif can be determined by, for example, gel shiftassay usually used by those skilled in the art. More specifically, thisassay can be carried out as follows: First, the detected DNA isincorporated into a vector so that its gene product forms a fusedprotein with GST and the vector is allowed to express the fused protein.The expression product is purified using GST as an indicator followed bymixing with a labeled DNA probe containing the GCN4 motif. This mixedsolution is analyzed by electrophoresis using nondenaturing acrylamidegel. Binding activity can then be evaluated based on the locations ofthe detected bands on the gel.

In addition, whether or not a certain DNA codes for a protein having thefunction of activating expression of rice seed storage protein can bedetermined by, for example, a reporter assay. More specifically, thisassay can be carried out as follows. First, a vector is constructed sothat a reporter gene is connected to and downstream of the promoter ofrice seed storage protein. This vector and a vector that expresses thegene product of a test DNA are introduced into the cells for thereporter assay, and the transcription activity of the test DNA geneproduct is evaluated by measuring the activity of the reporter geneproduct. An example of the promoter of rice seed storage protein thatcan be used for the reporter assay is the rice glutelin gene promoter.There are no particular restrictions to the reporter gene provided itsexpression can be detected, and any reporter gene that are usually usedin various assay systems by those skilled in the art, can be used. Apreferable example of the reporter gene is the β-glucuronidase (GUS)gene.

A DNA encoding a protein functionally equivalent to the RISBZ proteinset forth in SEQ ID NO: 2, 5, or 7 can be produced by, for example,methods well known to those skilled in the art including: methods usinghybridization techniques (Southern, E. M., Journal of Molecular Biology,Vol. 98, 503, 1975); and polymerase chain reaction (PCR) techniques(Saiki, R. K. et al. Science, 230, 1350–1354, 1985; Saiki, R. K. et al.Science, 239, 487–491, 1988). It is routine for a person skilled in theart to isolate a DNA with high homology to the RISBZ gene from rice andso forth using the RISBZ gene (SEQ ID NO: 1, 3, 4, or 6) or partsthereof as a probe, and oligonucleotides hybridizing specifically to thegene as a primer. Such a DNA encoding a protein functionally equivalentto the RISBZ protein, isolable by hybridization techniques or PCRtechniques, is included in the DNA of this invention.

Hybridization reactions to isolate such DNAs are preferably conductedunder stringent conditions. Stringent hybridization conditions of thepresent invention include conditions such as: 6 M urea, 0.4% SDS, and0.5×SSC; and those which yield a similar stringency to the conditions.DNAs with higher homology are expected to be isolated efficiently whenhybridization is performed under conditions with higher stringency, forexample, 6 M urea, 0.4% SDS, and 0.1×SSC. These DNAs isolated under suchconditions are expected to encode a protein having a high amino acidlevel homology with RISBZ protein (SEQ ID NO: 2, 5, or 7). Herein, highhomology means an identity of at least 50% or more, more preferablymeans an identity of at least 70% or more, and most preferably means anidentity of at least 90% or more (e.g., 95% or more) throughout theentire amino acid sequence. The degree of sequence identity can bedetermined by FASTA search (Pearson W. R. and D. J. Lipman Proc. Natl.Acad. Sci. USA. 85:2444–2448, 1988) or BLAST search.

The DNA of the present invention can be used, for example, to preparerecombinant proteins and to produce transgenic plants as describedabove.

A recombinant protein is usually prepared by inserting a DNA encoding aprotein of the present invention into an appropriate expression vector,introducing the vector into an appropriate cell, culturing thetransformed cells, and purifying expressed proteins. A recombinantprotein can be expressed as a fusion protein with other proteins so asto be easily purified, for example, as a fusion protein with maltosebinding protein in Escherichia coli (New England Biolabs, USA, vectorpMAL series), as a fusion protein with glutathione-S-transferase (GST)(Amersham Pharmacia Biotech, vector pGEX series), or tagged withhistidine (Novagen, pET series). The host cell is not limited so long asthe cell is suitable for expressing the recombinant protein. It ispossible to utilize, for example, yeast, plant, insect cells or variousother animal cells besides the above-described E. coli. A vector can beintroduced into a host cell by a variety of methods known to one skilledin the art. For example, a transformation method using calcium ions(Mandel, M. and Higa, A. Journal of Molecular Biology, 53, 158–162,1970;Hanahan, D. Journal of Molecular Biology, 166, 557–580, 1983) can beused to introduce a vector into E. coli. A recombinant protein expressedin the host cells can be purified and recovered from the host cells orthe culture supernatant thereof by known methods in the art. When arecombinant protein is expressed as a fusion protein with maltosebinding protein or other partners, the recombinant protein can be easilypurified via affinity chromatography.

The resulting protein can be used to prepare an antibody that binds tothe protein. For example, a polyclonal antibody can be prepared byimmunizing immune animals, such as rabbits, with a purified protein ofthe present invention or its portion, collecting blood after a certainperiod, and removing clots. A monoclonal antibody can be prepared byfusing myeloma cells with the antibody-forming cells of animalsimmunized with the above protein or its portion, isolating a monoclonalcell expressing a desired antibody (hybridoma), and recovering theantibody from the cell. The antibody thus obtained can be utilized topurify or detect a protein of the present invention. Accordingly, thepresent invention includes antibodies that bind to proteins of theinvention.

A plant transformant expressing DNAs of the present invention can becreated by inserting a DNA encoding a protein of the present inventioninto an appropriate vector, introducing this vector into a plant cell,and then, regenerating the resulting transformed plant cell.

On the other hand, a plant transformant in which the expression of theDNA of the present invention is suppressed can be created using a DNAthat suppresses the expression of a DNA encoding a protein of thepresent invention: wherein the DNA is inserted into an appropriatevector, the vector is introduced into a plant cell, and then, theresulting transformed plant cell is regenerated. The phrase “suppressionof expression of a DNA encoding a protein of the present invention”includes suppression of gene transcription as well as suppression oftranslation to protein. Furthermore, it also includes the completeinability of expression of DNA as well as reduction of expression.

The expression of a specific endogenous gene in plants can be suppressedby methods utilizing antisense technology conventional to the art. Eckeret al. were the first to demonstrate the antisense effect of anantisense RNA introduced by electroporation into plant cells by usingthe transient gene expression method (J. R. Ecker and R. W. Davis Proc.Natl. Acad. Sci. USA 83: 5372, 1986). Thereafter, the target geneexpression was reportedly reduced in tobacco and petunias by expressingantisense RNAs (A. R. van der Krol et al. Nature 333: 866, 1988). Theantisense technique has now been established as a means of suppressingtarget-gene expression in plants.

Multiple factors cause antisense nucleic acid to suppress thetarget-gene expression. These include the following: inhibition oftranscription initiation by triple strand formation; suppression oftranscription by hybrid formation at the site where the RNA polymerasehas formed a local open loop structure; transcription inhibition byhybrid formation with the RNA being synthesized; suppression of splicingby hybrid formation at the junction between an intron and an exon;suppression of splicing by hybrid formation at the site of spliceosomeformation; suppression of mRNA translocation from the nucleus to thecytoplasm by hybrid formation with mRNA; suppression of splicing byhybrid formation at the capping site or at the poly(A) addition site;suppression of translation initiation by hybrid formation at the bindingsite for the translation initiation factors; suppression of translationby hybrid formation at the site for ribosome binding near the initiationcodon; inhibition of peptide chain elongation by hybrid formation in thetranslated region or at the polysome binding sites of mRNA; andsuppression of gene expression by hybrid formation at the sites ofinteraction between nucleic acids and proteins. These factors suppressthe target gene expression by inhibiting the process of transcription,splicing, or translation (Hirashima and Inoue, “Shin Seikagaku JikkenKoza (New Biochemistry Experimentation Lectures) 2, Kakusan (NucleicAcids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression ofGenes),” Nihon Seikagakukai Hen (The Japanese Biochemical Society),Tokyo Kagaku Dozin, pp. 319–347, (1993)).

An antisense sequence of the present invention can suppress the targetgene expression by any of the above mechanisms. In one embodiment, if anantisense sequence is designed to be complementary to the untranslatedregion near the 5′ end of the gene's mRNA, it will effectively inhibittranslation of a gene. It is also possible to use sequencescomplementary to the coding regions or to the untranslated region on the3′ side. Thus, the antisense DNA used in the present invention includesa DNA having antisense sequences against both the untranslated regionsand the translated regions of the gene. The antisense DNA to be used isconnected downstream of an appropriate promoter, and, preferably, asequence containing the transcription termination signal is connected onthe 3′ side. The DNA thus prepared can be transfected into the desiredplant by known methods. The sequence of the antisense DNA is preferablya sequence complementary to the endogenous gene of the plant to betransformed or a part thereof, but it need not be perfectlycomplementary so long as it can effectively inhibit the gene expression.The transcribed RNA is preferably 90% or more, and most preferably 95%or more complementary to the transcribed products of the target gene.The complementary of sequences can be determined by the above-describedsearch methods. In order to effectively inhibit the expression of thetarget gene by means of an antisense sequence, the antisense DNA shouldbe at least 15 nucleotides long or more, preferably 100 nucleotides longor more, and still more preferably 500 nucleotides long or more. Theantisense DNA to be used is generally shorter than 5 kb, and preferablyshorter than 2.5 kb.

DNA encoding ribozymes can also be used to suppress the expression ofendogenous genes. A ribozyme means an RNA molecule that has catalyticactivities. There are many ribozymes having various activities. Researchon the ribozymes as RNA cleaving enzyme has enabled the design of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the M1RNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (MakotoKoizumi and Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Nucleic acid,Protein, and Enzyme) 35: 2191, 1990).

The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′side of C15 of the sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered important for theribozyme activity. It has been shown that the cleavage also occurs whenthe nucleotide at the 15th position is A or U instead of C (M. Koizumiet al. FEBS Lett. 228: 225, 1988). If the substrate binding site of theribozyme is designed to be complementary to the RNA sequences adjacentto the target site, one can create a restriction-enzyme-like RNAcleaving ribozyme which recognizes the sequence UC, UU, or UA within thetarget RNA (M. Koizumi et al. FEBS Lett. 239: 285, 1988; Makoto Koizumiand Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, andEnzyme), 35: 2191, 1990; M. Koizumi et al. Nucleic Acids Res. 17: 7059,1989). For example, in the coding region of the RISBZ gene (SEQ ID NO:1, 3, 4, or 6), there are pluralities of sites that can be used as theribozyme target.

The hairpin-type ribozyme is also useful in the present invention. Ahairpin-type ribozyme can be found, for example, in the minus strand ofthe satellite RNA of tobacco ringspot virus (J. M. Buzayan, Nature 323:349,1986). This ribozyme has also been shown to target-specificallycleave RNA (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751;Yo Kikuchi (1992) Kagaku To Seibutsu (Chemistry and Biology) 30: 112).

The ribozyme designed to cleave the target is fused with a promoter,such as the cauliflower mosaic virus ³⁵S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. If extra sequences have been added to the 5′ end or the 3′end of the transcribed RNA, the ribozyme activity can be lost. In thiscase, one can place an additional trimming ribozyme, which functions incis to perform the trimming on the 5′ or the 3′ side of the ribozymeportion, in order to precisely cut the ribozyme portion from thetranscribed RNA containing the ribozyme (K. Taira et al. (1990) ProteinEng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc. Natl. Acad.Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991) Nucleic AcidsRes. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res. 19: 5125).Multiple sites within the target gene can be cleaved by arranging thesestructural units in tandem to achieve greater effects (N. Yuyama et al.,Biochem. Biophys. Res. Commun. 186: 1271 (1992)). By using suchribozymes, it is possible to specifically cleave the transcriptionproducts of the target gene in the present invention, therebysuppressing the expression of the gene.

Endogenous gene expression can also be suppressed by co-suppressionthrough the transformation by DNA having a sequence identical or similarto the target gene sequence. “Co-suppression” refers to the phenomenonin which, when a gene having a sequence identical or similar to thetarget endogenous gene sequence is introduced into plants bytransformation, expression of both the introduced exogenous gene and thetarget endogenous gene becomes suppressed. Although the detailedmechanism of co-suppression is unknown, it is frequently observed inplants (Curr. Biol. 7: R793, 1997, Curr. Biol. 6: 810, 1996). Forexample, if one wishes to obtain a plant body in which the RISBZ gene isco-suppressed, the plant in question can be transformed via a vector DNAdesigned so as to express the RISBZ gene or DNA having a similarsequence to select a plant having the RISBZ mutant character, forexample, a plant with modified expression level of storage proteins inseeds, among the resultant plants. The gene to be used forco-suppression need not be identical to the target gene, but it shouldhave at least 70% or more sequence identity, preferably 80% or moresequence identity, and more preferably 90% or more (e.g., 95% or more)sequence identity. Sequence identity can be determined by using theabove-described search.

In addition, endogenous gene expression in the present invention canalso be suppressed by transforming the plant with a gene encoding aprotein having the dominant negative phenotype of the expression productof the target gene. “A DNA encoding a protein having the dominantnegative phenotype” as used herein means a DNA encoding a protein, whichupon expression, can eliminate or reduce the activity of the proteinencoded by endogenous gene inherent to the plant. An example thereof isa DNA that codes for a peptide having GCN4 binding ability and having notranscription activating domain of the protein of the present invention(for example, the peptide missing the 1st to 40th amino acids of theamino acid sequence of SEQ ID NO: 2 or a peptide of other proteinscorresponding thereto).

The vector used to transform plant cells is not particularly restrictedas long as it is capable of expressing an inserted gene in the cells.For example, a vector having a promoter for performing constitutive geneexpression in plant cells (e.g., the ³⁵S promoter of cauliflower mosaicvirus), or a vector having a promoter that is inductively activated byan external stimulus can be used. In addition, a promoter thatguarantees tissue-specific expression can also be suitably used.Examples of tissue-specific promoters include a promoter of glutelingene (Takaiwa, F. et al., Plant Mol. Biol. 17: 875–885, 1991) or apromoter of the RISBZ1 of the present invention for the expression inthe seeds of rice plants, and a promoter of glycinin gene for theexpression in the seeds of leguminous crops such as kidney beans, broadbeans and green peas or oil seed crops such as peanuts, sesame seeds,rape seeds, cottonseeds, sunflower seeds and safflower seeds, or apromoter of the major storage protein of each of the above crops such asa promoter of phaseolin gene in the case of kidney beans (Murai, N. etal., Science 222: 476–482, 1993) or a promoter of the gluciferrin genein the case of rape seed (Rodin, J. et al., Plant Mol. Biol. 20:559–563, 1992), a promoter of the patatin gene (Rocha-Sosa, M. et al.,EMBO J. 8: 23–29, 1989) for the expression in the root tuber ofpotatoes, a promoter of the sporamin gene for the expression in the roottuber of sweet potatoes (Hattori, T. and Nakamura, K., Plant Mol. Biol.11: 417–426, 1988), and a promoter of the ribulose-1,5-bisphosphatedecarboxylase gene for the expression in the leaves of spinach and othervegetables (Orozco, B. M. and Ogren, W. L., Plant Mol. Biol. 23:1129–1138, 1993).

The plant cell to which a vector is introduced used herein includesvarious forms of plant cells, such as cultured cell suspensions,protoplasts, leaf sections, and callus.

A vector can be introduced into plant cells by known methods, such asthe polyethylene glycol method, electroporation, Agrobacterium-mediatedtransfer, and particle bombardment. Plants can be regenerated fromtransformed plant cells by known methods depending on the type of theplant cell (Toki et al., (1995) Plant Physiol. 100:1503–1507). Forexample, transformation and regeneration methods for rice plantsinclude: (1) introducing genes into protoplasts using polyethyleneglycol and regenerating the plant body (suitable for indica ricecultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus Iand Spangenberg Eds., pp66–74); (2) introducing genes into protoplastsusing electric pulse, and regenerating the plant body (suitable forjaponica rice cultivars)(Toki et al (1992) Plant Physiol. 100,1503–1507); (3) introducing genes directly into cells by the particlebombardment, and regenerating the plant body (Christou et al. (1991)Bio/Technology, 9: 957–962); (4) introducing genes using Agrobacterium,and regenerating the plant body (Hiei et al. (1994) Plant J. 6:271–282); and so on. These methods are already established in the artand are widely used in the technical field of the present invention.Such methods can be suitably used for the present invention.

Once a transformed plant with the DNA of the present inventionintegrated into the genome is obtained, it is possible to gain progeniesfrom that plant body by sexual or vegetative propagation. Alternatively,plants can be mass-produced from breeding materials (for example, seeds,fruits, ears, tubers, tubercles, tubs, callus, protoplast, etc.)obtained from the plant, as well as progenies or clones thereof. Plantcells transformed with the DNA of the present invention, plant bodiesincluding these cells, progenies and clones of the plant, as well asbreeding materials obtained from the plant, its progenies and clones,are all included in the present invention. The plant body of the presentinvention is preferably a monocotyledon, more preferably a plant of thePoaceae, and most preferably a rice plant.

In addition, the present invention provides a plant body in which aforeign gene product has been highly expressed using the RISBZ gene ofthe present invention. The plant body of the present invention has inits genome a DNA construct in which the DNA of the present invention isoperably connected downstream of an expression control region, and a DNAconstruct in which a foreign gene is operably connected downstream of anexpression control region having a target sequence.

The DNA of the present invention or a foreign gene being “operablyconnected” downstream of an expression control region means that the DNAof the present invention or a foreign gene binds to an expressioncontrol region so as to induce the expression of the DNA of the presentinvention or a foreign gene by the binding of a transcription factor tothe expression control region.

The target sequence refers to a DNA sequence to which the RISBZ proteinof the present invention, which is a transcription factor, binds, and ispreferably a DNA sequence that contains the GCN4 motif or G/C box.Examples of the GCN4 motif include the sequences shown below which havebeen found in various genes:

* GCN4 Motif (name of gene containing GCN4 motif)

GCTGAGTCATGA/(GluB-1) SEQ ID NO: 8 CATGAGTCACTT/(GluA-1) SEQ ID NO: 9AGTGAGTCACTT/(GluA-3) SEQ ID NO: 10 GGTGAGTCATAT/(LMWG) SEQ ID NO: 11GGTGAGTCATGT/(Hordein) SEQ ID NO: 12 GATGAGTCATGC/(Gliadin) SEQ ID NO:13 AATGAGTCATCA/(Secalin). SEQ ID NO: 14

Preferable GCN4 motif sequences for use as target sequences include“GCTGAGTCATGA/SEQ ID NO: 8”, GATGAGTCATGC/SEQ ID NO: 13” and“AATGAGTCATCA/SEQ ID NO: 14”. Specific examples of a G/C box include thesequence, “AGCCACGTCACA/SEQ ID NO: 15”. Sequences in which the aboveGCN4 motif or G/C box is repeated in tandem are also included in thetarget sequence of the present invention, and a preferable example is asequence in which the GCN4 motif or G/C box are repeated in tandem fourtimes.

Examples of foreign genes include genes coding for antibodies, enzymes,and physiologically active peptides.

Moreover, the present invention provides a method of producing a plantbody in which a foreign gene product is highly expressed using the RISBZgene of the present invention. Examples of the methods for producing theplant body include a method of crossing “a plant body having a DNAconstruct in its genome, in which the DNA of the present invention isoperably connected downstream of an expression control region,” and “aplant body having a DNA construct in its genome, in which a foreign geneis operably connected downstream of an expression control region havingthe target sequence of the protein of the present invention.”

The above-described “DNA construct in which the DNA of the presentinvention is operably connected downstream of an expression controlregion,” and “the DNA construct in which a foreign gene is operablyconnected downstream of an expression control region having a targetsequence” can be introduced into the plant genome by a conventionalmethod by those skilled in the art, such as a method that uses theabove-mentioned agrobacterium.

In addition, crossing of plant bodies can be carried out by aconventional method for those skilled in the art. For example, in orderto prevent self-propagation, only the pollen is sterilized bydemasculating using the tip shearing method on the day of crossing or bydemasculating using hot water on the day of crossing to shake pollinatethe ear of the pollen mother.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing representing a genealogical tree based on thehomology of the amino acid sequence of RISBZ protein and O2-like bZIPprotein. The entire amino acid sequences of these proteins are comparedto understand the similarity and the evolutionary relationship of theseproteins.

FIG. 2 compares the amino acid sequences of RISBZ protein and O2-likebZIP protein. Outline letters on a black background shows the aminoacids that retained 50% or more. The presumed nuclear migration signal(NLSA: SV40-like motif) (Varagona, M. J. et al., Plant Cell 4:1213–1227, 1992) and the serine-rich phosphorylation sites are indicatedwith double lines and broken lines, respectively. The bold linesindicate the basic domain, which has a two-factor nuclear migrationsignal (NLSB) structure. Downward arrows indicate the leucine repeats.The primer used for the production of the rice bZIP probe was designedbased on the amino acid sequences indicated by rightward and leftwardarrows. BLZ1 (Vicente-Carbojos, J. et al., Plant J. 13: 629–640, 1998)and BLZ2 (Onate, L. et al., J. Biol. Chem. 274: 9175–9182, 1999)represent O2-like bZIP proteins isolated from barley, O2 (Hartings, H.et al., EMBO J. 8: 2795–2801, 1989) and OHP1 (Pysh, L. D. et al., PlantCell 5: 227–236, 1993) from maize, SPA from wheat (Albani D. et al.,Plant Cell9: 171–184, 1997), O2-sorg from sorghum (Pirovano, L. et al.,Plant Mol. Biol. 24: 515–523, 1994), and O2-coix from adlay (Vettore, A.L. et al., Plant Mol. Biol. 36: 249–263, 1998).

FIG. 3 is a continuation of FIG. 2.

FIG. 4 shows the structure of a gene that codes for O2-like bZIPprotein. The structures of the intron/exon region of the BLZ1 gene ofbarley and the Opaque2 gene of maize (O2) (Hartings, H. et al., EMBO J.8: 2795–2801, 1989), sorghum (O2-sorg) and adlay (O2-coix) are shown.The thick bars and thin lines represent exons and introns, respectively.The numbers indicate the number of nucleotides of the exons and introns.

FIG. 5 is a photograph representing the result of a Northern blotshowing the transcription patterns of the RISBZ genes. Northern blottinganalysis was performed on the whole RNA extracted from the root,seedling, and maturing seeds (5, 10, 15, 20, and 30 DAF) using a uniquenucleotide sequence of a region downstream of the bZIP domain for theprobe. In order to compare transcription patterns, the analysis was alsoconducted using the. GluB-1 gene-coding region as the probe. The stainedimages of 25S rRNA obtained using ethidium bromide are shown as acontrol.

FIG. 6 represents the results of histological analysis of the RISBZ1promoter/GUS reporter gene in a transformed rice plant.

(A) is a schematic drawing of the RISBZ1 promoter/GUS reporter gene. (a)and (b) show the sequence from the −1674^(th) to +4^(th) nucleotidescounting from the transcription initiation point of the RISBZ1 gene andthe sequence from the −1674^(th) to +213^(th) gene that contains uORF,respectively, both connected to the GUS reporter gene on a binaryvector. (c) shows the GluB1 promoter (−245 to +18) sequence binding tothe GUS reporter gene on a plasmid vector.

(B) are photographs showing the expression of GUS reporter gene in aseed during the maturation process. After cutting the seed (10 DAF) of arice plant, into which the reporter gene was introduced, in thelongitudinal direction, the cut seed was immersed in X-gluc solution andincubated at 37° C. EN indicates the endosperm, while EM indicates theembryo.

(C) is a graph showing the GUS activity of a seed extract of atransformed rice plant. 15 DAF seeds were used for analysis. Thepromoter structures of the introduced genes are as shown in (a) and (b)of (A), respectively. Vertical lines indicate the mean value. MUrepresents 4-methylumbelliferone.

FIG. 7 shows photographs of gel electrophoretic patterns as determinedfrom a methylation interference experiment for identifying the RISBZ1protein-binding site on the GluB1 promoter. Each of the strands (top andbottom) of the promoter fragment of the GluB1 gene (−245 to +18) waslabeled. After partially methylating each strand, they were incubatedwith GST-RISBZ1 protein, the fragments that did not bind to the proteinand the fragment that bound to the protein were each collected andsubjected to electrophoresis after chemically cleaved by piperidine. Thesites (indicated by asterisks) that were not cleaved by piperidine wereonly found in the GCN4 motif.

FIG. 8 shows the result of electrophoresis in gel shift analysis toinvestigate the binding capability of RISBZ1 protein to the GCN4 motif.

(A) shows 21-bp DNA fragments that contain the GCN4 motif of aWILD:GluB-1 promoter sequence (−175 to −155) of an oligonucleotide usedas the probe and competitor. M1 to M7 are a series of 21-bp DNAfragments that were mutated every 3 bp. The GCN4 motif is underlined.

(B) through (F) show the results of gel shift analysis of the GST-RISBZfused protein. A 21-bp DNA fragment (WILD) was added as the probe. (B)is for GST-RISBZ1, (C) for GST-RISBZ2, (D) for GST-RISBZ3, (E) forGST-RISBZ4, and (F) for GST-RISBZ5. The competitor was added to astoichiometric ratio of 100 times or more against the probe. Lane 1: Noprotein; Lane 2: No competitor; and Lanes 3 to 10: With Competitor (wildtype (W) and M1 to M7).

FIG. 9 represents heterodimer forming ability of RISBZ1 with other RISBZproteins.

(A) shows the vector structure used as the in vitrotranscription/translation reaction template. The vectors contain DNAcoding for full-length RISBZ1 protein, short-form RISBZ2 protein(sRISBZ2: 218 to 329), or short-form RISBZ3 protein (sRISBZ3: 126 to237).

(B) shows photographs of gel electrophoretic patterns representing theresults of a DNA binding assay. In lanes 2, 4, 6, and 8, DNA complexesthat bound to the full length or short-form protein were detected. Inlanes 3 and 7, DNA complexes that bound to the heterodimer of fulllength RISBZ1 protein and short-form protein were detected.

FIG. 10 shows the results of identification of thetranscription-activating domain determined by transient analysis.

(A) shows the structure of the reporter and effector plasmid. A GUS genein which 9 copies of GAL4-DNA binding sites and CaMV35S core promotersequence are linked was used for the reporter. The effector plasmidcontained DNA coding for a protein in which the GAL4 DNA binding domainwas linked to the N-terminal side of truncated RISBZ1 protein.

(B) is a graph showing GUS activity when the reporter and effectorplasmid were used.

FIG. 11 shows the hydropathy patterns of the N-terminal region of RISBZ1(WT) and mutant RISBZ1 (M1 to 8) proteins determined by the formula ofKyte and Doolittle (Kyte, J. and Doolittle, R. F. J., Mol. Biol. 157:105–132, 1982). Positive values indicate hydropathy.

FIG. 12 schematically shows the transcription activity measurementsystem of RIZBZ1 using GUS activity as the indicator, photographs ofNorthern blot analysis, and a graph showing GUS activity measurementresults. The ordinate of the graph represents GUS activity that is theindicator of the strength of the transcription activity of eachtranscription factor.

FIG. 13 is a graph showing the recognition sequences of transcriptionfactors RISBZ1, Opaque2, SPA, and RISBZ3 (RITA1). The ordinate of thegraph represents GUS activity that is the indicator of the strength ofthe transcription activity of each transcription factor. The sequencesused in the experiment are shown below the graph.

FIG. 14 is a graph showing the transcription activating ability of theRISBZ1 of the present invention relative to GCN4 motifs originating invarious genes. The ordinate of the graph represents GUS activity, whichis the indicator of the strength of the transcription activity of eachtranscription factor. The nucleotide sequences of the GCN4 motifs usedin the experiment are shown below the graph.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below withreference to Examples, but is not to be construed as being limitedthereto.

EXAMPLE 1

Isolation of cDNA Clones Encoding the bZIP Transcription Factor FromSeed cDNA Libraries

Fourteen-day leaves and roots of rice plant (Oryza sativa L. c. v.Mangetumochi) cultivated by hydroponics were frozen in liquid nitrogenand kept at ˜80° C. until use. Maturing rice seeds were collected fromrice plants cultivated in the fields.

Using oligonucleotide primers designed from highly conserved amino acidsequences (SNRESA and KVKMAED) within the bZIP domain of the Opaque 2(O2)-like protein, RT-PCR was performed by using poly(A)⁺ mRNA as atemplate, which was prepared from the rice seeds. From poly (A) RNAextracted from seeds at 6 to 16 days after flowering (DAF) (Takaiwa F.et al. Mol. Gen. Genet. 208: 15–22, 1987), single-stranded cDNA wassynthesized by reverse transcription using oligo(dT)₂₀ as a primer andSuperscript reverse transcriptase (Gibco BRL, Paisly, UK). Next, cDNAwas amplified using a pair of primers (5′-TCC AAC/T A/CGI GAA/G A/TCIGC-3′; SEQ ID NO: 16, and 5′-GTC CTC C/TGC CAT CTT CAC CTT-3′; SEQ IDNO: 17). These primers were designed based on highly conserved aminoacid sequences within the bZIP-type transcription factors that wereexpressed in cereal seeds. After dissolving the single-stranded cDNA ina PCR reaction mixture containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂,50 mM KCl, 0.01% (w/v) gelatin, 200 μM dNTPs, 1 μM oligonucleotideprimers, TaqI polymerase was added to the mixture and the resultingmixture was incubated in a thermal cycler at 94° C. for 5 min. cDNA wasthen synthesized and amplified by three-cycle PCR (for 1 min at 94° C.,for 1 min at 40° C., and then for 2 mins at 72° C.) followed by 30-cyclePCR (for 1 min at 94° C., for 1 min at 55° C., and then for 2 mins at72° C.). The amplified DNA fragment was cloned into a TA cloning vector(pCR2.1; Invitrogen), and subjected to sequencing by using the ABI PRISMdye terminator sequence system. The reaction products were analyzed byABI PRISM 310 Genetic Analyzer (Perkin Elmer-Applied Biosystems) todetermine the nucleotide sequences of at least 50 clones. The obtainednucleotide sequence data was analysed and searched on databases by usingthe GENETYX and BLAST algorisms. As a result, five distinct DNAfragments with 213-bp were found. Two of these were identical to thebZIP domain sequences of REB (Izawa T. et al. Plant Cell 6: 1277–1287,1994) and the RITA1 (Nakase M. et al. Plant Mol. Biol. 33: 513–522,1997). Using the five DNA fragments with 213-bp as primers, a cDNAlibrary was prepared from RNA of maturing (6-16DAF) seeds (ZAPII;STRATAGENE) This was then screened to obtain their full-length cDNAscorresponding to each of the fragments under high stringent conditions.[α-³²P]-dCTP was incorporated into the DNA fragments by random priming(Amersham Pharmacia Biotech) and the resulting fragments were used asprobes. As a pre-hybridisation solution, a mixture containing 5×SSC, 5×Denhard's solution, 0.1% SDS, 50% formamide, 100 μg/ml salmon sperm DNAwas used. After hybridization, filters were washed once at 55° C. with amixture consisting of 2×SSC and 0.1% SDS, and then twice at 55° C. witha mixture consisting of 0.1×SSC and 0.1% SDS.

Based on the homologies to each nucleotide sequence, the cDNA clonesobtained were termed as RISBZ1 (rice seed b-Zipper 1) (SEQ ID NO: 1),RISBZ2, RISBZ3, RISBZ4 (SEQ ID NO: 4), and RISBZ5 (SEQ ID NO: 6). Amongthem, RISBZ2 and RISBZ3 were identical to REB (Izawa T. et al. PlantCell 6: 1277–1287, 1994) and RITAL (Nakase M. et al. Plant Mol. Biol.33: 513–522, 1997), respectively, which have previously been isolatedfrom cDNA libraries of seeds and leaves.

EXAMPLE 2

Identification of RISBZ cDNA

The newly identified RISBZ cDNAs (RISBZ1, RISBZ4, and RISBZ5) werecharacterized in detail as described below. RISBZ1 cDNA was the longest,which had 1742 bp in length excluding poly(A), and contained a readingframe encoding 436 amino acids that had 46,491 Dal of an estimatedmolecular weight. RISBZ4 and RISBZ5 have reading frames encoding 278 and295 amino acids; their estimated molecular weights are 29,383 Dal and31,925 Dal respectively.

RISBZ1 mRNA has a longer leader sequence (245 bases long) than averageleader sequences. Interestingly, a small open reading frame, encoding 31amino acid residues, was found within the leader sequence in theupstream of the actual initiation codon of the RISBZ1 protein. Similarsmall upstream open reading frames (UORF) have previously been found inmaize Opaque 2 (O2) (Hartings H. et al. EMBO J. 8: 2795–2801, 1989),wheat SPA (Albani D. et al. Plant Cell 9: 171–184, 1997), and barleyBLZ1 and BLZ2 (Vincente-Carbojos J. et al. Plant J. 13: 629–640, 1998;Onate L. et al. J. Biol. Chem. 274: 9175–9182, 1999), but these uORFshave little homology with each other. It has previously been reportedthat uORF of the maize O2 mRNA is involved in translational control.uORF was found only in RISBZ1 mRNA but not in other RISBZ mRNA.

The flanking sequence of the initiation codon is GCAATGG. This sequencecoincided with eukaryotic translational initiation sequence, c(a/c)(A/G) (A/C)cAUGGCG, derived from monocotyledonous plants. There were 100bps between the initiation codon and uORF. The open reading frameencoding RISBZ1 had two identical termination codons (TAG). There were229 bps between the termination codon and poly (A) sequence. Thepolyadenylation signal sequence (AATATA) was found in the region at −19to −24 from the site to which poly(A) was added.

RISBZ1 is closely related to rice REB (Nakase M. et al. Plant Mol. Biol.33: 513–522, 1997), maize OHP-1 and OHP-2 (Pysh L. D. et al. Plant Cell5: 227–236, 1993), and barley BLZ1 (Vincente-Carbojos J. et al. Plant J.13: 629–640, 1998) (FIG. 1), and showed the homologies of 48.2% (riceREB), 45.7% (barley BLZ1), and 46.6% (maize OHPL), respectively, at theamino acid level. Furthermore, these bZIP domains were highly conserved(73.7% to 76.3%). At the amino acid level, the homologies of RITA1(RISBZ3) with RISBZ4 and RISBZ5 were 88.8% and 47.6% respectively. Bycontrast, the homology of RISBZ4 with RISBZ5 was 48.2%. RISBZ3, RISBZ4,and RISBZ5 comprise a unique group among the O2-like transcriptionfactors that were previously reported. Furthermore, the five RISBZ cDNAsisolated from the seed cDNA library could be classified into two groupsbased upon the amino acid homology (FIG. 1). The RISBZ3, RISBZ4, andRISBZ5 lacked the N- and C-terminal regions present in RISBZ1 andRISBZ2, and their sizes reduced about 100 to 150 amino acid residuescompared with those of RISBZ1 and RISBZ2 (FIGS. 2 and 3).

RISBZ1 and RISBZ2 were rich in proline residues at their N-terminalregion, which lacked in other RISBZ proteins (FIGS. 2 and 3). RISBZ1 andRISBZ2 were also rich in acidic amino acids at the peripheral region ofthe 60^(th) amino acid residue from their N-termini and at theintermediate region located in the upstream of their bZIP domains. Theseproline-rich or acidic amino acid-rich regions were found in otherO2-like transcription factors.

Since serine-rich sequence (SGSS) was found in the region ranging from207^(th) to 210^(th) residues of RISBZ1, the protein was considered tobe a target sequence of casein kinase II (Hunter T. and Karin M. Cell70: 375–387, 1992) (FIGS. 2 and 3) Similar sequence (SSSS) was alsofound in RISBZ2. However, it was missing in the other RISBZ proteins(FIGS. 2 and 3).

So far, two nuclear transition signals (NLSA: an SV-40-like motif andNLSB: a 2-factor motif) have been identified, which are involved intransport of maize Opaque2 (O2) proteins from cytoplasm into nucleus(Varagona M. J. et al. Plant Cell 4: 1213–1227, 1992). These motifs weresearched on RISBZ1 and sequences homologous to NLSA and NLSB were foundat the same sites as O2 (101 to 135 and 232 to 264).

EXAMPLE 3

Genomic Structure of the RISBZ1 Gene

Using primers designed from the nucleotide sequence of the RISBZL cDNA,the genomic region encoding promoter and RISBZL protein was isolated.The PCR reaction was performed using rice genomic DNA as a template andtwo pairs of oligonucleotide primers (RIS1f:5′-ATGGGTTGCGTAGCCGTAGCT-3′/SEQ ID NO: 18 and RELr5:5′-TTGCTTGGCATGAGCATCTGT-3′/SEQ ID NO: 19) and (RELf2:5′-GAGGATCAGGCCCATAT-3′/SEQ ID NO: 20 and RIS1r:5′-TCGCTATATTAAGGGAGACCA-3′/SEQ ID NO: 21). DNA fragments were amplifiedusing TAKARALA Taqpolymerase (TAKARA) in a thermal cycler through30-cycle reactions for 10 sec at 98° C., for 30 sec at 56° C. and for 5min at 68° C. The promoter region of the RISBZ1 gene was also amplifiedby thermal asymmetric interlaced (TAIL) PCR, based on the method by Liuet al, in which three oligonucleotides were used as specific primers,tail1: 5′-TGCTCCATTGCGCTCTCGGACGAG-3′/SEQ ID NO: 22, tail2:5′-ATGAATTCGCGAGGGGTTTTCGA-3′/SEQ ID NO: 23, and tail3:5′-GTTTGGGAGAAATTCGATCAAATGC-3′/SEQ ID NO: 24.

The results revealed that the RISBZ1 gene comprises of six exons andfive introns (FIG. 4). The constitution of exon/intron in this RISBZ1gene was identical to that of the maize O2 (Hartings H. et al. EMBO J.8: 2795–2801, 1989), Sorghum O2 (Pirovano L. et al. Plant Mol. Biol. 24:515–523, 1994), adlay O2 (Vettore A. L. et al. Plant Mol. Biol. 36:249–263, 1998), and barley BLZ1 (Vicente-Carbojos J. et al. Plant J. 13:629–640, 1998) genes (FIG. 4).

The transcription initiation site of the RISBZ1 gene was determined bythe primer extension analysis according to the method of Sambrook et al.(Sambrook J. et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., pp.7.79–7.83, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). Specifically, a primer, 5′-ATGGTATGGTGTTCCTAGCACAGGTGTAGC-3′ (SEQID NO: 25), was produced by labelling with T4 kinase, the 5′ end of theoligonucleotide comprising 30 nucleotides, which was complementary to asequence immediately downstream of a desired region. Reversetranscription reaction was conducted using this primer and 5 μg of mRNAas a template, and a Superscript reverse transcriptase kit (Gibco BRL,Paisly, UK). This reaction was carried out in a mixture comprising 20 mMTris-HCl, 50 MM MgCl₂, 10 mM DTT, 500 μM dNTP, 100,000 cpm primer, 5 μgmRNA, and 200-unit Superscript reverse transcriptase (Gibco BRL, Paisly,UK), for 50 min at 42° C.

As a result, the transcription initiation site was mapped to the 245-ntupstream region from the translation initiation codon of the RISBZ1gene. A ‘TATA’ box was localized at −30 to −35-nt from the transcriptioninitiation site. Three ‘ACGT’ motifs were found in the 63-, 123-, and198-bp upstream regions from the transcription initiation site but noneof motifs responsible for expression of seed-specific genes, such as,GCN4 and ‘AACA’ were found. In contrast, a number of the recognitionsequences for Dof domain protein, ‘AAAG’, were found. These motifs maybe involved in stage- and/or tissue-specific expression of the RISBZ1gene. For example, if the ‘ACGT’ motif is a target sequence of theRISBZ1 protein, the RISBZ1 gene may be autoregulated by itself. However,when the RISBZ1 promoter/GUS reporter gene and the ³⁵S CaMVpromoter/RISBZ1 gene were introduced into protoplast cells, notranscriptional activation of the reporter gene was observed. These datasuggest that the RISBZ1 promoter has no target sequence for the RISBZ1protein; namely, the ‘ACGT’ motif found in the RISBZ1 promoter is not atarget sequence of the protein. Therefore, the RISBZ1 gene is probablynot autoregulated. In contrast, upon overexpression of the rice prolaminbox binding factor (RPBF) gene (which recognizes the Dof domain)transcription of the RISBZ1 promoter/GUS reporter gene is activated.This suggests that the recognition sequences of the Dof domain proteinsare involved in specific expression of the RISBZ1 gene.

EXAMPLE 4

Tissue-Specificity of the RISBZ mRNA

Northern blotting was carried out to analyze the expression of the RIABZgene. According to the method by Takaiwa et al. (Varagona M. J. et al.Plant Cell 4: 1213–1227, 1992), total RNA was extracted from 5 to 30 DAFseeds, roots, and seedling (5-, 10-, 15-, 20- and 30-DAF), and wastransferred to membrane filters after fractionation by agarose gelelectrophoresis. As probes, the following DNA fragments ranging from thedownstream sequence of the bZIP domain-encoding region to the 3′non-coding region in the RISBZ cDNA were used: RISBZ1, 354-bp rangingfrom 1388^(th) to 1742^(nd) nucleotides; RISBZ2, 346-bp ranging from1351^(st) to 1696^(th) nucleotides; RISBZ3, 486-bp ranging from 741^(st)to 1226^(th) nucleotides; and RISBZ5, 621-bp ranging from 742^(nd) to1362^(nd) nucleotides.

Hybridization was carried out in a solution containing 5×SSC, 5×Denhard's solution, 0.1% SDS, and 50% formamide, at 45° C. After thehybridization, the membrane filters were washed twice for 30 min with amixed solution comprising 2×SSC and 0.1% SDS, and then twice for 30 minwith a mixture comprising 0.1×SSC and 0.1% SDS.

As shown in FIG. 5, the RISBZ1 gene was expressed only in seeds, not inother tissues analyzed. The largest amount of the RISBZ1 mRNA wasaccumulated in seeds harvested from 5 DAF to 10 DAF. Such a highaccumulation of mRNA was maintained until 15 DAF, and graduallydecreased towards maturing. The peak of the RISBZ1 gene expressionappeared at an earlier stage than that of the glutelin gene. Theglutelin mRNA expression was detected from 5 DAF, had a peak at 15 DAF,and was then gradually decreased (FIG. 5). This result suggests that theRISBZ1 acts as an activator of the glutelin gene. Similar expressionpatterns have also been reported in the maize O2 (Hartings H. et al.EMBO J. 8: 2795–2801, 1989), wheat SPA (Albani D. et al. Plant Cell 9:171–184, 1997), and barley BLZ2 genes (Onate L. et al. J. Biol. Chem.274: 9175–9182, 1999).

The RISBZ2 was expressed in all the tissues analyzed. The RISBZ3 andRISBZ 4 were expressed specifically in seeds at later stages of maturing(FIG. 5). The RISBZ3 and RISBZ 4 mRNA levels gradually increased until20DAF and then decreased. The expression level of RISBZ5 was extremelylow, compared with other RISBZ genes, and its mRNA peak was at 10 DAF.

EXAMPLE 5

Expression of the RISBZ1 Promoter/GUS Reporter Gene Construct inTransformants

To examine an expression pattern of the RISBZ1 gene, the sequencefragment ranging from −1674 to +213 nt numbering from the transcriptioninitiation site, was ligated upstream of GUS gene. This reporter genewas introduced into rice plant by using Agrobacterium (FIG. 6A).Transformed rice plant (Oryza sativa L. c. v. kitaake) was constructedas follows. Two oligonucleotide primers with the PstI or BamHIrestriction site at its 5′ end, 5′-AAAACTGCAGTTTTCTGA-3′ (SEQ ID NO: 26)and 5′-AATGGATCCGCGAGGGGTTTTCGAA-3′ (SEQ ID NO: 27), were used toamplify the 5′-end regions (from −1674^(th) to +4^(th) and from−1674^(th) to +213rd) of the RISBZ1 gene by PCR. The PCR reaction wascarried out in a reaction mixture (10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂,50 mM KCl, 0.01% (w/v) gelatine, 200 μM dNTPs, 1 μM primers, 0.5 μgtemplate DNA, and 2.5-unit TaqI polymerase) by 30 cycles of incubationfor 1 min at 94° C., for 1 min at 50° C. and for 2 min at 72° C. Afterdigestion with restriction enzymes, PstI and BamHI, the PCR product wascloned into the plasmid vector pBI201, and was cleaved with restrictionenzymes, PstI and SacI. The resulting DNA fragment containing the RISBZ1promoter/GUS gene was inserted between the Sse8387I and SacI sites ofthe binary vector p8cHm, which contains the CaMV35S promoter/hygromycinphosphotransferase (HPT) gene. Transformation was performed according tothe method described in Goto F. et al. Nature Biotech. 17: 282–286.

The reporter plasmid was constructed as follows. 1×21 bp, 3×21 bp, and5×21 bp of GCN4 motifs/GUS genes, as constructed by Wu et al. (Wu C. Y.et al. Plant J. 14: 673–683, 1998), were used as the reporter. A pair of48-bp oligonucleotides with overhanged (ACGT) 5′ ends, which werecomplementary to each other, was associated to construct tetramerscomprising 12-bp wild-type GCN4 motif (GCTGAGTCATGA/SEQ ID NO: 8) andmutant GCN4 motif (GCTTCCTCATGA/SEQ ID NO: 28). These double-strandedoligonucleotide were inserted into the SalI and StuI sites of the−46CaMV/GUS reporter gene.

Transient assay for rice callus protoplast was carried out according tothe method described by Wu et al. The GUS activity was measuredaccording to the method of Jefferson (Jefferson R. A. Plant Mol. Biol.Rep. 5: 387–405, 1987), by measuring fluorescence intensity of4-methyl-umbelliferone derived from the glucuronide precursor. Using BioRad Kit, the concentration of proteins was measured. Bovine serumalbumin was used as a standard protein.

As shown in FIG. 6B, high GUS activities was observed in the aleulon andsub aleulon layers of maturing seeds, but not in germs. The GUS activitywas not detected in roots, leaves, and stems even by highly sensitivefluorescence measurement. These results indicate that the RISBZ1 gene isexpressed exclusively in the aleulon and sub aleulon layers. To examinethe role of the 5′-end untranslated region and uORF, the GUS activitywas compared with that of a plant, which lacked uORF ranging from−1674^(th) to +4^(th) numbering from the transcription initiation site(FIG. 6A). As a result, no change in the expression site was observeddue to the lack of uORF (FIG. 6B), but 5- to 10-fold weaker promoteractivities were observed (FIG. 6C). These data suggest that the 5′untranslated region may play a role in upregulation of the translation,in contrast to the results in the maize O2 in which uORF functions as asuppressor of the translation (Lohmer S. et al. Plant Cell 5: 65–73).

EXAMPLE 6

Transcription Activating Ability of Five RISBZ Proteins Through TheirBinding to the GCN4 Motif

Transcription activating ability of the five RISBZ proteins throughtheir binding to the GCN4 motif was measured by transient assay. Theplasmids, into which each RISBZ1 protein-encoding sequences were ligateddownstream of CaMV35S promoter as an effector, were prepared. Effectorplasmids were prepared as follows. The plasmid that encodes RISBZ1lacking its N-terminal region was prepared by PCR. In order to amplifycDNA encoding the regions ranging from 41^(st), 81^(st), 121^(st), and161^(st) amino acids numbering from the N-terminus of RISBZ1 to itsC-terminus the following primers were designed:

Forward primers RIS1-1: 5′-AACCATGGTGCTGGAGCGGTGCCCGT-3′ (SEQ ID NO: 29)RIS1-2: 5′-AACCATGGCGGCGGAGGCGGCGGCG-3′ (SEQ ID NO: 30) RIS1-3:5′-CCCCATGGAGTACAACGCGATGC-3′ (SEQ ID NO: 31) RIS1-4:5′-AACCATGGTTGGTTCCATCCTGAGT-3′ (SEQ ID NO: 32) RIS1-5:5′-AACCATGGCTCATGCCAAGCAAGCT-3′ (SEQ ID NO: 33) RIS1-6:5′-AACCATGGATGAAGAAGATAAAGTGAAG-3′ (SEQ ID NO: 34) Reverse primerBRIS1R: 5′-TAGGATCCGCTCCTACTACTGAAGCT-3′. (SEQ ID NO: 35)

These primers were designed to have an NcoI or BamHI restriction site attheir 5′ end. Since a translational initiation codon was lost bydeletion of its N-terminal region, ATG of the NcoI restriction site wasutilized. cDNAs were amplified by PCR comprising incubation for 2 min at94° C., 30-cycle reaction for 1 min at 94° C., for 1 min at 50° C., andfor 2 min at 72° C., followed by incubation for 5 min at 72° C. The PCRproducts were digested with restriction enzymes, NcoI and BamHI, andthen purified through agarose gel electrophoresis. The purified cDNAfragments were finally inserted into the pRT100 vector (Topfer R. et al.Nucl. Acids Res. 15: 5890, 1987).

Plasmids encoding the fusion proteins comprising GAL4 DNA-binding domain(amino acid residues from 1^(st) to 147^(th)) and the RISBZ1 or RISBZ2gene were also constructed. In order to amplify the cDNA region encodingvarious N-terminal region of RISBZ1 and RISBZ2 by PCR using Pfu Taqpolymerase (STRATAGENE), the following reverse primers, to which a BamHIsite, a terminal codon, and an SstI site were added at its 5′-end, wereprepared as well as the following forward primers:

Forward primers RISBZ1-F1: 5′-AAGGATCCAATGGAGCACGTGTTCGCC-3′ (SEQ ID NO:36) RISBZ1-F2: 5′-AAGGATCCGGCGGCGGAGGCGGCGCG-3′ (SEQ ID NO: 37)RISBZ1-F3: 5′-GCCGGATCCAGTTGGTTCCATCCTGAG-3′ (SEQ ID NO: 38) RISBZ1-F4:5′-AAGGATCCTGATGAAGAAGATAAAGT-3′ (SEQ ID NO: 39) RISBZ1-F1-2:5′-AAGGATCCAGGAGTAGATGACGTCGGC-3′ (SEQ ID NO: 40) RISBZ1-F1-3:5′-AAGGATCCAGACGAGATCCCCGACCCGCT-3′ (SEQ ID NO: 41) Reverse primersRISBZ1-R1: 5′-TAGAGCTCTACGCCGCCGGCATCGGGCT-3′ (SEQ ID NO: 42) RISBZ1-R2:5′-TAGAGCTCTAAAGGATCATATTTCCCAT-3′ (SEQ ID NO: 43) RISBZ1-R1-1:5′-TAGAGCTCTAGGCGGCCGCCGCCGGCTG-3′ (SEQ ID NO: 44) RISBZ1-R1-2:5′-TAGAGCTCTACGGCGGCGGCGGAGCCCA-3′. (SEQ ID NO: 45)

cDNAs encoding various N-terminal regions of RISBZL and RISBZ2 wereamplified by PCR comprising incubation for 2 min at 94° C., 30 cycles ofreaction for 1 min at 94° C., for 1 min at 50° C., and for 1 min at 72°C., and then incubation for 5 min at 72° C., using the above-describedprimers. The amplified cDNAs were digested with BamHI and SacIrestriction enzymes, and were purified by 2% agarose gelelectrophoresis. The purified cDNA fragments were ligated downstream ofthe GAL4 DNA domain-encoding region in the ³⁵S-564 vector digested withthe same restriction enzymes so that their reading frames were matched.Mutations were also introduced into the N-terminal regions of RISBZ1 byPCR mutagenesis. The cDNA sequences were confirmed, and their partialsequence from 1^(st) to 57^(th) amino acid residues was amplified byPCR. The products were ligated downstream of the GAL4 DNAdomain-encoding region in their reading frames.

In addition, reporter plasmids, into which the GUS gene, and one orthree repeat(s) of the 12-bp GCN4 motif or one or five repeat(s) of the21-bp GCN4 motif were inserted, were constructed. For negative controlexperiments, a reporter plasmid comprising four repeats of a mutant12-bp GCN4 motif and the GUS reporter gene was used. The mutant 12-bpGCN4 motif has a mutation in the target sequence that is recognized bythe RISBZ1 and O2. These plasmid constructs were introduced alone or incombination with other reporter or effector plasmid into rice protoplastcells prepared from its callus culture, and the GUS activity wasassayed. When the reporter plasmid or effector plasmid was introducedalone into the protoplast, the GUS activity was detected at a low level.As shown in Table 1, however, in the presence of ³⁵S/RISBZ1 or ³⁵S/O2,which were introduced as effector plasmids, the transcription of thereporter gene was activated. Even in the presence of these effectorplasmids, the transcriptional activity of the GUS gene downstream of themutant 12-bp GCN4 motif was the same level as that of background. Theseresults indicate that the RISBZ1 gene product activates the reportergene mediated by the GCN4 motif. The transcriptional activity of thereporter gene induced by the RISBZ1 gene product was slightly higherthan that induced by the O2 gene product. As shown in Table 2, theactivity induced by RISBZ1 was enhanced depending on the copy number ofthe GCN4 motif. 1 to 12 copies of 21-bp GCN motif were assayed, and thetranscriptional activity was enhanced proportionately up to 9 copies.However, even though the other RISBZ genes were expressed under thecontrol of the ³⁵S CaMV promoter, the transcriptional activity of thereporter gene was less than or equal to 1.4% of that induced by theRISBZ1 or O2 gene product. Thus, it was revealed that only the RISBZ1protein can activate the transcription through its binding to the GCN4motif.

TABLE 1 Effector GUS activity (pM 4-MU/min/mg protein) 35S/Opaque2 2658± 318 35S/RISBZ1 2994 ± 157 35S/RISBZ2 44 ± 7 35S/RISBZ3  1.3 ± 1.235S/RISBZ4 17.3 ± 0.9 35S/RISBZ5   31 ± 8.8

The 4×12-bp GCN4 motifs/GUS reporter gene was introduced into protoplastcells together with the effector plasmid, and the GUS activity wasmeasured. Data were obtained from three independent measurements.

TABLE 2 Effector GUS Activity (pM 4-MU/min/mg protein) Reporter (−) (+)RISBZ1 (+) Opaque2 1 × 12-bp GCN4 32 ± 1.5 295 ± 4.5  182 ± 6  (9.2*)(5.6*) 4 × 12-bp GCN4 21  604 ± 24.5 452 ± 7.5  (28.7*) (21.5*) 1 ×21-bp GCN4 30 ± 3   1318 ± 55.5  1139 ± 22.5  (43.9*) (37.9*) 5 × 21-bpGCN4 104 13222 ± 1094  11932 ± 22.5   (127.1*) (114.7*)As a reporter, the 1×12-bp, 4×12-bp, 1×21-bp or 5×21-bp GCN4 motif/GUSgene was used. This table shows the GUS activity induced by theexpression of RISBZ1 (+RISBZ1) gene or by Opaque2 (+Opaque2) gene.

EXAMPLE 7

Binding Site of the RISBZ1 Protein

The present inventors have previously discovered that the O2 proteinrecognizes the GCN4 motif (TGAGTCA) that is present in the promoterregion ranging from −165^(th) to −160^(th) of GluB-1, a glutelin gene(Wu C. Y. et al. Plant J. 14: 673–683, 1998). By a methylationinterference experiment, the present inventors have also determined thebinding site of the RISBZ1 protein in the promoter region of the GluB-1gene.

Production and purification of the GST-RISBZ1 fusion protein wereperformed as follows. Five coding regions from RISBZ1 cDNA wereamplified by PCR using oligonucleotide primers to which the followingappropriate restriction enzyme sites were added at their 5′ end;BamHI-blunt ends for RISBZ1, BamHI-XhoI for RISBZ2, BamHI-SalI forRISBZ3, BamHI-SalI for RISBZ4, and BamHI-XhoI for RISBZ5. Afterdigestion with the restriction enzymes, the PCR products were ligatedinto the cloning sites of the pGEX-4T-3 vector (Amersham PharmaciaBiotech). The GST-RISBZ fusion protein was expressed according to themethod of Suzuki et al. (Suzuki A. et al. Plant Cell Physiol. 39:555–559, 1998). After affinity purification, the GST fusion protein wasdialyzed against a binding buffer comprising 20 mM HEPES-KOH pH 7.9, 50mM KCl, 1 mM EDTA, and 10% glycerol, for four hours, and immediatelystored at −80° C.

Methylation interference experiment was performed as described byWeinberger et al. (Weinberger J. et al. Nature 322: 846–849, 1986). The5′-flanking region (from −245^(th) to +18th nucleotides) of the GluB1gene was digested with restriction enzymes, SalI and BamHI, and the endsof the fragment was labeled with [α-³²P] dCTP by a ‘fill-in’ reaction.The labelled fragment was methylated by treating it with dimethylsulphate, mixed with GST-RISBZ1, and then incubated. Usingnon-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis, the DNAfragment complexed with GST-RISBZ1 and free DNA fragments were separatedfrom each other. These DNA fragments were further purified by DEAESepharose column chromatography, were treated with piperidine, and werefractionated by 6% denaturing acrylamide gel electrophoresis.

As shown in FIG. 7, the GST-RISBZ1 fusion protein protected guanineresidues that locate in the −165^(th) to −160^(th) region of the GluB-1promoter. The guanine residues protected were the same residuesprotected in the O2 promoter (Albani D. et al. Plant Cell 9: 171–184,1997). A guanine residue present in the ‘ACGT’ motif (also termed as anA/G hybrid box) at the −79^(th) to −76^(th) residues in the promoterregion ranging from −197^(th) to +18th, was not protected.

Furthermore, gel shift assay was conducted as described below to examinewhether the RISBZ1 protein can recognize the GCN4 motif.

A pair of oligonucleotides complementary to each other, which wasprepared by adding TCGA sequence was added to 21-nt fragment of GluB1promoter region (from −175^(th) to −155^(th)), was labeled at its endswith [α-³²P] dCTP by ‘fill-in’ reaction for use as a probe. Seven pairsof complementary oligonucleotides with mutations every three contiguousnucleotides (FIG. 8A) were also synthesized for use as mutant competitorfragments and were annealed. Gel shift analysis using the GST fusionprotein was carried out by a method described by Wu et al. (Wu C. Y. etal. Plant J. 14: 673–683, 1998) and by Suzuki et al. (Suzuki A. et al.Plant Cell Physiol. 39: 555–559, 1998). The labeled oligonucleotideprobe was mixed with 0.5 μg of the GST-RISBZ fusion protein, andincubated for 20 min at room temperature. In competition experiments,the competitor fragment was added to the mixture at the 100-fold orhigher molecular weight ratio. The reacted mixture was analyzed bynon-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis.

The detection of shift bands showed that the GST-RISBZ1 protein was ableto bind to the 21-bp DNA fragment containing the GCN4 motif (FIG. 8B).Furthermore, as shown FIG. 8A, the 21-bp DNA fragments with mutation inevery three contiguous nucleotides were used as competitors andexamined. When the DNA fragments with the mutations in the GCF motifwere added as the competitor, the binding of the DNA fragments that wereadded as probes was hardly or not inhibited at all (FIGS. 8B to F). Bycontrast, when the DNA fragments with mutations in the franking sequenceof the GCN4 motif were added as the competitor, the shift bandsdisappeared (FIGS. 8B to F). Since the mutation of the GCN4 motifmarkedly affects the binding of the RISBZ1 protein to the motif, it wasrevealed that the RISBZ1 protein recognizes the GCN4 motif sequencespecifically. The similar experiments carried out using the other RISBZproteins revealed that all the RISBZ proteins could specificallyrecognize the GCN4 motif. As shown in FIGS. 8B to F, the affinity ofeach RISBZ proteins for the GCN4 motif slightly varies. In the cases ofRISBZ2 and RISBZ5, when the DNA fragments with mutations in the frankingsequence of the GCN motif were used as the competitor, the shift bandswere not disappeared completely (FIGS. 8C and F).

From these results, it was revealed that the RISBZ proteins specificallyrecognize the GCN4 motif with slightly variable affinities.

EXAMPLE 8

Ability of RISBZ1 Protein to Form a Heterodimer

It was considered that the RISBZ1 protein, a bZIP-type transcriptionfactor, binds to the GCN4-like motif upon forming a heterodimer withother RISBZ proteins. Therefore, the ability of RISBZ1 to heterodimerizewith RISBZ2 or RISBZ3 was examined. The full-length RISBZ1 protein, andshort-form-RISBZ2 protein (sRISBZ2) and short-form RISBZ3 protein(sRISBZ3) were prepared using wheat germ extracts (FIG. 9A), and wereused for DNA binding assay. The in vitro translation was carried out asfollows. The coding region of RISBZ1 cDNA and the bZIP domain-encodingregions of RISBZ2 cDNA and RISBZ3 cDNA were amplified using thefollowing forward primers with the NcoI site at their 5′ ends andreverse primers encoding a terminator codon and the BamHI site;

For RISBZ1 R1F: 5′-AAACCATGGAGCACGTGTTCGCCGT-3′ and (SEQ ID NO: 46)BRIS1r: 5′-TAGGATCCGCTCCTACTACTGAAGCT-3′; (SEQ ID NO: 47) For sRISBZ2dR2-1: 5′-AAACCATGGAGGGAGAAGCTGAGACC-3′ and (SEQ ID NO: 48) R2ra1:5′-AAAGGATCCTACATATCAGAAGCGGCGGGA-3′; and (SEQ ID NO: 49) For sRISBZ3,dR3-1: 5′-AAACCATGGATATAGAGGGCGGTCCA-3′ and (SEQ ID NO: 50) R3ral:5′-AAAGGATCCTACAGCCCGCCCAGGTGGCCG-3′. (SEQ ID NO: 51)

PCR amplification was carried out in a reaction mixture comprising 10 mMTris-HCl pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.01% (w/v) gelatine, 200 μMdNTPs, 1 μM primers, 0.5 mg template DNA, and 2.5-unit TaqI polymeraseby 30 cycles of incubation for 1 min at 94° C., for 1 min at 50° C. andfor 2 min at 72° C.

The PCR products were digested with restriction enzymes, NcoI and BamHI,and were ligated into the pET8c cloning vector (Novagen) to constructplasmids. Using these plasmids as templates, in vitrotranscription/translation (TNT coupled wheat germ extract systems;Promega) was performed for the production of the full-length RISBZ1protein, and short-form-RISBZ2 (RISBZ2s) and -RISBZ3 (RISBZ3s). For gelshift assay, 4 μl of the wheat germ extract that was used in the abovereaction was used.

Gel shift assay was employed to separate homodimers and heterodimersbound to the 21-bp GCN4 motifs. After pre-incubating RISBZ1 with sRISBZ2and sRISBZ3, the DNA probes comprising the GCN4 motif were added to theincubation mixture. The results indicate that RISBZ homodimers as wellas heterodimers can bind to the GCN4 motif. Therefore, it wasdemonstrated that the RISBZ proteins form heterodimers with the othermembers of the RISBZ family.

EXAMPLE 9

Involvement of the N-Terminal Region of the RISBZ1 Proteins in theTranscriptional Activation

Transient assay was performed to identify the domain of the RISBZ1protein involved in transcription activation. The GUS gene, to whichthree copies of the 21-bp GCN4 motif and the core promoter sequence ofCaMV35S were connected, was prepared as a reporter. Various domains ofthe RISBZ1 proteins were expressed using the CaMV35S promoter in orderto examine if these domains can activate the reporter gene.

A series of effector plasmids encoding RISBZ1 proteins in which every 40amino acids from N-terminus to the basic domain were deleted (encodingthe amino acids region ranging from 41^(st) to 436^(th), 81^(st) to ₄₃₆,121^(st) to 436^(th), 161^(st) to 436^(th), 201^(st) to 436^(th), or235^(th) to 436^(th) in the amino acid sequence set forth in SEQ ID NO:2), were constructed. When the effector plasmid encoding the full-lengthRISBZ1 protein and the reporter plasmid (the GUS gene to which fourcopies of the 12-bp GCN motif and the core promoter sequence of CaMV35Swere linked) were introduced into protoplasts, approximately 30-foldhigher activity of GUS was detected compared to that of protoplast intowhich the reporter plasmid alone was introduced. When thetranscriptional activity of this reporter gene was set as 100%, theactivity of the gene with deletion of the first 40-amino acid wasdecreased to 20%. Furthermore, the activity of the reporter gene wasdecreased gradually to 10% by deleting each 40 amino acids. Hence, itwas suggested that the N-terminal 40 amino acid residues of RISBZ1 aremainly involved in the transcription activation.

To further analyze the association of the N-terminal 40 amino acids ofRISBZ1 with its transcription activating ability, various fusionproteins between the DNA binding domain of the yeast transcriptionalactivating factor GAL4 and various portions of the RISBZ1 protein wereconstructed and expressed for the gain-of-function assay. As shown inFIG. 10, a plasmid, in which the coding sequences of fused proteinscomprising the GAL4-DNA binding domain and various regions of RISBZ1were connected downstream of the CaMV35S promoter, was constructed andused as an effector. These effector plasmids were introduced intoprotoplast together with a reporter construct (the GUS gene, to whichnine copies of the GAL4-DNA binding site and CaMV35S core promoter wereconnected).

The significant difference was not found in transcription activatingability of the fusion protein comprising the GAL4-DNA binding domain andthe partial amino acid sequence from 1st to 235^(th) amino acids ofRISBZ1, compared with that of a series of the fused proteins in whichamino acids were deleted towards the 27^(th) residue from the C-terminalresidue of RISBZ1 (FIG. 10). The transcription activating ability of thefusion protein with the first 20 amino acid residues were dramaticallydecreased (FIG. 10). A fusion protein with deletion of the N-terminaleight residues of RISBZ1 lost the transcriptional activity. In contrast,fusion proteins comprising the GAL4-DNA binding domain and other regionof RISBZ1 (from 27^(th) through 57^(th), 81^(st) through 234^(th),161^(st) through 234^(th), or 235^(th) through 436^(th) in SEQ ID NO: 2)had no effect on the transcriptional activity of the reporter gen. Theseresults suggest that the proline-rich domain within the N-terminal 27amino acid residues of the RISBZ1 protein, rather than the acidicdomain, involves in the transcription activation.

EXAMPLE 10

Difference Between RISBZ1 and Other RISBZ Proteins in TranscriptionActivating Ability Analyzed by Domain Swapping

Although all the members of the RISBZ protein family have similaraffinity for the GCN4 motif sequence, only the RISBZ1 has thetranscription activating ability. To find out the reason of thisdifference, domain swapping between RISBZ1-, and RISBZ2- orRISBZ3-protein was carried out. The N-terminal region at 1^(st) through299th of RISBZ1, which resides upstream of the bZIP domain, was replacedwith the N-terminal region, 1^(st) through 229^(th) of RISBZ2 or 1^(st)through 137^(th) of RISBZ3.

Fusion proteins that have the N-terminal region of RISBZ1 together withthe DNA binding domain of RISBZ2 or RISBZ3 showed only approximately 15%or 38% of the transcription activating ability, respectively, comparedwith that of the full-length RISBZ1. In contrast, fusion proteins thathave the N-terminal region of RISBZ2 or RISBZ3 together with the RISBZ1DNA binding domain showed a slightly higher transcription activity thanthat induced by the RISBZ1 DNA binding domain alone.

These results indicate that the N-terminal region is mainly involved inthe transcription activation. The lower level of the RISBZ2 or RISBZ3transcription activating ability may be due to deletion or mutation ofthe region corresponding to RISBZ1 transcription activating domainduring evolution. Alternatively, the formation process of transcriptionactivating domain may be responsible for that. It is highly possiblethat the lower activity of RISBZ3 is due to the lack of the proline-richdomain present in RISBZ1. This applies to RISBZ4 and RISBZ5. The resultsof the gel shift assay probably exclude the possibility that thedifferences of affinity with GCN4 motif raise the differences oftranscription activating ability.

The proline-rich domain of RISBZ1 was also highly conserved in RISBZ2,but the transcription activating ability of RISBZ2 was extremely lowcompared to that of RISBZ1. When an effector plasmid that encodes afused protein comprising the N-terminal 27 amino acid residues of RISBZ2including proline-rich domain and the GAL4-DNA binding domain wasintroduced together with a reporter plasmid encoding the GCN4 motifconnected to the GUS gene into protoplast, no increased activity of GUSwas observed.

Since only eight-residue differences among the N-terminal 27 residueswere observed between RISBZ1 and RISBZ2, the present inventors haveexamined which of the residues among the eight are responsible for thedifference in transcription activating ability. The eight amino acidresidues of RISBZ1, which were different from RISBZ2, were replaced oneby one with the residues of RISBZ2, and the resulting chimericN-terminal sequences comprising 40 amino acids were fused with theGAL4-DNA binding domain to construct effector plasmids encoding thefused proteins. These effector plasmids were introduced into protoplasttogether with the reporter plasmid in which the GCN4 motif was fusedwith the GUS gene. Among eight effector plasmids, all the effectorconstructs, except for those encoding a protein with replacement of theseventh residue counting from the N-terminus of RISBZ1, did not activatethe transcription of the reporter gene. It was presumed using the Kyteand Doolittle formula that all these seven substitutions of amino acids,which were lost transcription activating ability, would induce thechange of a hydropacy pattern (FIG. 11).

EXAMPLE 11

Use of the Transcription Factor RISBZ1 for Plant Breeding

The present inventors have examined the possibility to use thetranscription factor, RISBZ1, which has a transcription activatingability for plant breeding. In order to specifically overexpress thetranscription factor in seeds, rice-plants were transformed with aplasmid construct that encodes the RISBZ1 gene under the control of thepromoter of the rice prolamin gene, which encodes a seed storageprotein, with 13-kDa molecular masses. The DNA fragment ranging from theEcoRI site, located at the −29^(th) position, to the poly (A) additionsite of the RISBZ1 gene was linked to the prolamin promoter encompassingfrom the −652^(nd) through −13^(th) from the translation initiation siteATG of the gene. The construct was inserted into the binary pGTV-Barvector, and the resulting vector was introduced into rice plants usingAgrobacterium. By this approach, 28 independent transformed lines wereestablished. Screening of rice plants that overexpress the RISBZ1 mRNAwas carried out by Northern hybridization of RNA extracted from maturingseeds using cDNA of RISBZ1 as a probe (FIG. 12). These linesoverexpressing RISBZ1 were crossed with the transformed rice plants, inwhich a plasmid construct encoding five tandem repeats of the 21-bp GCN4motif (5′-GTTTGTCATGGCTGAGTCATG-3′/SEQ ID NO: 52), a target of theRISBZ1 protein, linked to the minimum promoter/GUS reporter had beenintroduced.

As a result, it was revealed that the expression level of GUS reportergenes were, due to overexpression of RISBZ1 enhanced by 400-times ormore (450 to 750 nmol/min/mg protein) than that of controls, 5×GCN4lines (11 to 14 nmol/min/mg protein) (FIG. 12). These results suggestthat the transcription of foreign genes can be highly activated byconnecting the foreign genes downstream of the target sequence of thetranscription factor RISBZ1 with transcription activating ability andoverexpressing RISBZ1.

The RISBZ1 proteins can activate not only the glutelin gene but alsoother storage protein genes. The ³⁵S CaMV promoter/RISBZ1 fusionconstruct together with the glutelin promoter/GUS, glutelin promoter(−980^(th) to ATG)/GUS, or 13-Kd prolamin promoter (from −652^(nd) to−29th)/GUS, was introduced into rice protoplast using electroporation,and the transient expressions of them were examined.

The results indicated that the RISBZ1 protein bound to the targetsequences containing GCN4 motifs in these promoters and activated thetranscription of the foreign genes. It was revealed that thetranscriptions were activated 5 to 10-fold in the case of the 13-Kdprolamin promoter and 20 to 30-fold in the case of the globulinpromoter, higher than that of the background. Therefore, methylationinterference reaction was used to determine how RISBZ1 recognizes thenucleotide sequences of these genes.

The results showed that three GCN4 motifs (TGACACA/SEQ ID NO:86,GATGACTCA/SEQ ID NO:87, and TGACTCAC/SEQ ID NO:88) of the prolamin geneand three motifs different from the GCN4 motif (GGTGACAC SEQ ID NO:89,GTATGTGGC /SEQ ID NO:90, and GATCCATGTCAC/SEQ ID NO:91) of the globulingene were recognized by the RISBZ1 protein. To determine specificsequences in the promoters that are recognized by the RISBZ1, transientexpression of the GUS gene was examined by using a chimeric promotersequence in which the G, A, C, G/C, MG, or C/A box, GCN4, 22-Kd zeinbinding site and four repeats of 12-bp sequence including the b-32binding site were inserted in tandem into the 46 CaMV 35S corepromoter/GUS reporter gene. The results indicate that the RISBZ1 proteinpreferentially recognizes the G/C box and GCN4 motif (FIG. 13).

Furthermore, it was studied to see if the RISBZ1 protein recognizedvarious distinct GCN4 motifs present in the promoter for the storageprotein genes. The results indicate that the flanking sequences of thecore sequence ‘TGAGTCA’ of GCN4 motif influence transcription activatingability, and that the GCN4 motifs of the wheat gliadin gene and ryesecalin gene have high transcription activating ability (FIG. 14).

INDUSTRIAL APPLICABILITY

The present invention provides novel transcription factors that regulatethe expression of rice seed storage proteins, and genes that encode thetranscription factors. It is expected that the expression of many seedstorage proteins regulated by the RISBZ1 protein of the presentinvention having transcription activating ability can be enhanced byintroducing the gene encoding the RISBZ1 protein into cells tooverexpress it. The present invention also provides novel geneexpression systems in which a useful foreign gene, encoding such as anantibody and an enzyme, can be highly expressed using the transcriptionfactor of the present invention, by linking the recognition sequence ofthe transcription factor, the GCN4 motif, in tandem and introducing itinto the promoter for a gene encoding a seed storage protein tofacilitate its binding to the transcription factor. Thus, expression ofthe gene encoding storage protein and the useful foreign gene can begreatly enhanced under the control of the modified promoter. Thisenables abundant accumulation of a seed storage protein in endosperm,and more nutritious seeds (e.g. rice) and production of seeds in whichuseful proteins are highly accumulated.

1. An isolated nucleic acid molecule comprising: a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or b) a DNA encoding SEQ ID NO:2.
 2. The nucleic acid molecule of claim 1, which encodes a protein that binds to the GCN4 motif or activates expression of a rice seed storage protein.
 3. The nucleic acid molecule of claim 1, which is derived from a rice plant.
 4. A vector comprising the nucleic acid molecule of claim
 1. 5. An isolated nucleic acid molecule encoding an antisense RNA complementary to the full-length transcription product of a nucleic acid sequence, and wherein said nucleic acid sequence comprises: a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or b) a DNA encoding SEQ ID NO:2.
 6. A method of producing a protein, comprising the steps of culturing a transformed cell, and collecting the protein expressed from said transformed cell or from the supernatant of said transformed cell, and wherein said protein comprises: a) the amino acid sequence encoded by SEQ ID NO:1, or SEQ ID NO:3; or b) the amino acid sequence of SEQ ID NO:2.
 7. A transformed cell comprising a nucleic acid sequence comprising: a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or b) a DNA encoding SEQ ID NO:2.
 8. The transformed cell of claim 7, which is a plant cell.
 9. A plant which is derived from the transformed cell of claim 8, wherein said plant comprises said nucleic acid sequence.
 10. A plant derived from a progeny of, or a clone of the plant of claim 9, wherein said derived plant comprises said nucleic acid sequence.
 11. A reproductive material of the plant of claim 9, wherein said reproductive material comprises said nucleic acid sequence.
 12. A reproductive material of the plant of claim 10, wherein said reproductive material comprises said nucleic acid sequence.
 13. A plant which comprises a) a first nucleic acid construct comprising; i) a first expression control region, and ii) a first nucleic acid sequence downstream of said first expression control region, wherein said first nucleic acid sequence comprises: A) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or B) a DNA encoding SEQ ID NO:2; and b) a second nucleic acid construct comprising; i) a second expression control region having the target sequence of a protein encoded by said first nucleic acid sequence, and ii) a foreign nucleic acid sequence downstream of said second expression control region.
 14. The plant of claim 13, wherein said target sequence comprises the GCN4 motif.
 15. The plant of claim 14, wherein said GCN4 motif comprises the sequence as set forth in SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14.
 16. The plant of claim 13, wherein said target sequence comprises a G/C box.
 17. A method of producing the plant of claim 13, wherein said method comprises a step of crossing a first plant with a second plant, wherein said first plant comprises: a) a first nucleic acid construct comprising; i) a first expression control region, and ii) a first nucleic acid sequence downstream of said first expression control region, wherein said first nucleic acid sequence comprises: A) the coding region of a DNA as set forth in SEQ ID NO:1, or SEQ ID NO:3; or B) a DNA encoding SEQ ID NO:2; and wherein said second plant comprises: b) a second nucleic acid construct comprising; i) a second expression control region having the target sequence of a protein encoded by said first nucleic acid sequence, and ii) a foreign nucleic acid sequence downstream of said second expression control region.
 18. An isolated nucleic acid encoding a protein having a sequence at least 95% identical to SEQ ID NO:2, wherein said protein binds to the GCN4 motif and activates expression of a rice seed storage protein. 